Abnormal energy regulation may contribute to onset and progression of chronic metabolic conditions such as obesity, diabetes mellitus, cardiovascular disease, and cancer, which cause approximately 60% of the world's mortality [rev. in (1). Our lab is interested in how long chain fatty acids (LCFAs) reach nuclei, bind peroxisomal proliferator receptor alpha (PPARa), and initiate transcription for energy metabolism or storage. We hypothesize that liver fatty acid binding protein (L-FABP) transfers and channels LCFAs to nuclei, binding to and initiating PPARa transcriptional activity. Using purified L-FABP and PPARa, L-FABP overexpressed L- cells, L-FABP null mice, and cultured hepatocytes from L-FABP null mice, we propose to: Aim 1. Determine if the phenotype of L-FABP null mice is consistent with abnormal PPARa regulation. L-FABP null mice share many PPARa null mouse features (inhibition of LCFA oxidation, hypertriglyceridemia, sex/age-dependent obesity). L-FABP null mice exhibit upregulation of SCP-2/SCP-x, the converse of upregulation of L-FABP in SCP-2/SCP-x null mice. We have bred SCP-x, SCP-2/SCP-x, and L-FABP/SCP- 2/SCP-x null mice to clarify the in vivo role of LCFA/LCFA-CoA binding proteins in PPARa regulation. Aim 2. Examine the role of L-FABP in targeting LCFAs to the nucleus for interaction with PPARa. L-FABP enhances saturated LCFA targeting to nuclei. We will use novel fluorescent polyunsaturated (n-3, n-5) and branched-chain (phytanic acid) LCFAs, fluorescent-L-FABP (EYFP-, Cy3-, Cy5-) and immunogold EM, to show if: (i) L-FABP cotransports bound LCFAs into nuclei;(ii) LCFAs enhance L-FABP distribution into nuclei;(iii) nuclear targeting of LCFAs depend on relative binding affinities of L-FABP and PPARa. Aim 3. Resolve molecular interactions of L-FABP with PPARa. Physical and immunological techniques show that L-FABP binds PPARa in vitro. We will examine ligand specificity, conformational responsiveness, and co-activator or co-repressor binding, using: (i) purified proteins in vitro;(ii) FRET between EYFP-L- FABP/ECFP-PPARa or Cy3-L-FABP/Cy5-PPARa in living cells;(iii) immunogold EM;and (iv) fluorescence correlation spectroscopy in living cells (L-cells, primary cultured hepatocytes). Aim 4. Determine the mechanism of L-FABP-mediated LCFA transfer to PPARa. We will determine if LCFA transfers from L-FABP to PPARa by direct molecular interactions or by LCFA diffusion. These findings will contribute to our basic understanding of how different types of fatty acids may activate a nuclear receptor, and thereby induce transcription of genes directing their energy metabolism and storage. Differences in this nuclear receptor/fatty acid energy regulation could contribute to the pathogenesis of obesity, insulin resistance, type 2 diabetes mellitus, and hyperlipidemic conditions.